Background: Successful generation of patient-derived organoids (PDOs) from pancreatic ductal adenocarcinoma (PDAC) depends on an adequate amount of viable tissue. After routine endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) yields tissue for pathological diagnosis, additional needle passes are typically required to obtain organoid-culture material. This study examined whether air-flush tissue recovery (ATR) of residual needle-tract tissue can derive pancreatic cancer organoids without extra passes. Methods: We enrolled 10 PDAC patients who underwent EUS-FNA at two centers (The Ninth Medical Center of Chinese PLA General Hospital and the Second Hospital of Hebei Medical University) between February and June 2026. Using a self-controlled design, conventional FNA tissue strips (FNA group) and air-flush-recovered needle-tract residuals (ATR group) were collected from each patient and used to establish organoids. The two groups were compared for P3-generation passage rate, histological features, and molecular characteristics. Results: The ≥P3 success rate was 60.0% (6/10) in both the FNA and ATR groups. The overall organoid establishment rate (at least one method) was 80.0% (8/10). Hematoxylin and eosin (HE) staining and immunohistochemistry showed that organoids from both sources retained the histological morphology and key molecular features of the primary tumor, with consistent expression patterns. Conclusions: Harvesting residual tissue from the EUS-FNA needle tract by air flushing enables successful establishment of pancreatic cancer PDOs. The establishment success rate is comparable to that of organoids derived from conventional FNA specimens. In high-risk patients who cannot tolerate additional passes, a single ATR sample can still provide material for organoid generation; simultaneously culturing FNA tissue strips together with ATR residuals improved the overall organoid establishment rate from one puncture. This minimally invasive protocol is straightforward to perform and merits validation in larger cohorts.